To elucidate the regulatory mechanism of the acid tolerance response (ATR) in Bifidobacterium longum BBMN68, this study investigated the role of response regulator BBMN68＿47 in mediating ATR through genomic and proteomic approaches. An overexpression strain of BBMN68＿47 with a 3 × FLAG tag was constructed via molecular cloning, and the recombinant plasmids (pDP152-BBMN68＿47, pDP152-3 × FLAG, and pDP152-3 × FLAG-BBMN68＿47) were validated through polymerase chain reaction (PCR), sequencing, and Western blot. Under weak acid induction (pH 4.5), chromatin immunoprecipitation sequencing (ChIP-seq) identified 118 high-confidence DNA binding peaks, over 80% of which were localized at transcription start sites (TSS). Bioinformatics analysis predicted seven candidate DNA binding motifs. Electrophoretic mobility shift assay (EMSA) analysis further confirmed specific binding of BBMN68＿47 to two conserved motifs: CTGGGCGTTT(Probe 0235) and CTGGACGAATCCTG(Probe 0075), regulating the expression of its own gene BBMN68＿47(autoregulatory) and the S-adenosylmethionine synthetase (metK) gene, respectively. These findings establish BBMN68＿47 as a central transcriptional regulator in B. longum ATR, orchestrating key acid-resistant gene networks through conserved and novel DNA motifs. This work offers a theoretical basis for elucidating the molecular mechanism of the ATR of B. longum and engineering stress resistance in probiotics.